Primary cancer cells exhibit heterogeneity in clonogenicity, the capacity to proliferate and form colonies in vitro. The cancer stem cell (CSC) model accounts for this heterogeneity by proposing that each cancer consists of a small population of cells capable of unlimited growth and self-renewal, known as CSCs, and a much larger population of cells, descendants of the CSCs, that have lost self-renewal capacity and are undergoing terminal differentiation. Evidence supporting this model has been reported for several malignancies including acute myelogenous leukemia, brain cancer and breast cancer. The CSC model has important implications for cancer therapy; eradication of CSCs, the cells responsible for maintenance of the neoplasm, would be necessary and sufficient to achieve cure.
Myelodysplastic syndrome (MDS) is a clonal disorder of haematopoietic tissue, characterized by peripheral blood cytopenias, apoptosis of bone marrow haematopoietic progenitors, abnormal blood cell morphology (dysplasia) and a marked propensity to evolve into acute leukemia. The central paradox of MDS biology resides in the observation that the MDS clone, which is characterized by reduced numbers of mature progeny and by maturing progenitors that exhibit impaired clonogenicity and a high rate of apoptosis, nonetheless comes to dominate the bone marrow at the expense of residual normal haematopoiesis and thereby causes disease. The cancer stem cell model suggests a resolution to this paradox, namely that the MDS clone, despite the defects seen in its differentiating members, out-competes normal haematopoiesis because of a selective advantage at the stem cell level. It is hypothesized that this competitive advantage consists in an increased capacity of MDS stem cells for self-renewal.
The natural history of MDS is highly heterogenous, with some cases causing chronic cytopenias and others rapidly progressing to acute leukemia. Patients diagnosed with MDS have a life expectancy of 6 months to 5 years, and despite the recent development of some promising new therapies that offer hope for a small subset of patients with MDS, the mainstay of treatment for this disease remains supportive for palliative care with blood transfusion. Thus, most patients diagnosed with MDS face the prospect of a shortened life expectancy, impaired quality of life because of dependency on transfusions, and dread and uncertainty regarding the onset of acute leukemia.
Acute leukemia (AL) is an aggressive cancer of the blood forming cells in the bone marrow. It may arise secondary to preexisting hematopoietic conditions such as MDS, or de novo. Despite the many advances made in the understanding of leukemia biology over the past three decades, therapy for AML remains, in most cases, debilitating and ineffective. Further progress in improving the efficacy of anti-leukemia therapy hinges upon the identification of methods that allow for the targeting of the leukemia stem cell. Leukemia is a disease characterised by impairment of differentiation. Leukemia stem cells are the culprit of the disease. These rare cells (<1% of the population) are the only leukemia cells that are immortal. These cells are responsible for the initiation and maintenance of the leukemia. Eradication of the leukemia stem cell therefore, would be necessary and sufficient for cure. The rest of the leukemia cells in an AML patient are non-stem leukemia cells, these comprise the vast majority of the patient's leukemia cell burden. Non-stem leukemia cells are “benign” cells that either have a finite ability to divide or have lost the ability to divide altogether. Non-stem leukemia cells arise from the differentiation of leukemia stem cells. In contrast to current therapies that target both leukemia stem and non-stem cells, differentiation therapy aims at inhibiting the ability of leukemia stem cells to self-renew and inducing the differentiation of leukemia stem cells into non-stem leukemia cells. Differentiation therapy promises to be much more effective, selective and less toxic than chemotherapy.
NR2F6, known also as EAR-2, is an orphan nuclear receptor and a member of the chicken ovalbumin upstream promoter (COUP) family of nuclear receptors. The nuclear receptors (NRs) comprise a very large family of ligand activated transcription factors. Multiple lines of evidence suggest a role for NR signalling in the transcriptional regulation of haematopoiesis. Acute promyelocytic leukemia is invariably associated with gene fusions involving the retinoic acid receptor α (RARα) and one of five different partners, PML, PLZF, NPM, NuMA, and STAT5b. Patients with this disease respond to treatment with the RARα ligand, all trans retinoic acid (ATRA). Dominant negative mutants of RARα enhance mast cell development and reduce granulocyte and macrophage development in multipotential haematopoietic cell lines, and also block myeloid development in transduced murine bone marrow. Although targeted disruption of RARα in the mouse has little effect on haematopoiesis, in vitro studies revealed an increased proportion of morphologically immature granulocytes in RARα1/RARγ double mutants. In addition to this, in vitro studies suggest a role for the thyroid hormone receptor in erythropoiesis and for the PPARγ in monocyte/macrophage development. A role for the vitamin D receptor in myeloid differentiation is suggested by 1,25-dihydroxyvitamin D3-induced terminal differentiation and cell cycle arrest of a variety of leukaemic cell lines. Although little is known of the downstream genes regulated by NRs in haematopoiesis, evidence suggests that the cdk inhibitor p21 and the transcription factor C/EBPε may be targets of RARα in myelopoiesis.
NR2F6, known also as EAR-2, is an orphan nuclear receptor that was cloned in a search for homologues of the retroviral oncogene v-erbA using low stringency hybridization (see Miyajima, N., et al., (Identification of two novel members of erbA superfamily by molecular cloning: the gene products of the two are highly related to each other. Nucleic Acids Res, 16 (23): p. 11057-74. 1988)). EAR-2 is a member of the chicken ovalbumin upstream promoter (COUP) family of nuclear receptors. The COUPs function in vitro as transcriptional repressors, antagonizing the activation ability of a wide range of nuclear receptors that play prominent roles in differentiation. Accordingly, aberrant expression of COUP-TFI inhibits retinoid-induced epithelial and neuronal differentiation in vitro (Please see Kyakumoto, S., M. Ota, and N. Sato (Inhibition of retinoic acid-inducible transcription by COUP-TFI in human salivary gland adenocarcinoma cell line HSG. Biochem Cell Biol, 77 (6): p. 515-26. 1999), Neuman, K., et al., (Orphan receptor COUP-TF I antagonizes retinoic acid-induced neuronal differentiation. J Neurosci Res, 41 (1): p. 39-48. 1995) and Adam, F., et al., (COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells. Mol Endocrinol, 14 (12): p. 1918-33. 2000)). The roles of COUP-TFI and COUP-TFII in mammalian development have been studied by targeted deletion in the mouse. COUP-TFI deficient mice exhibit numerous defects in axonal development, including failure of development of the nucleus of the 9th cranial nerve. COUP-TFII deletion causes widespread defects in angiogenesis and cardiac development, leading to embryonic lethality in mid-gestation. Seven-up (svp), the Drosophila COUP family homologue, is also important in embryonic development; with null mutations of seven-up being embryonic lethal. svp is involved in decisions of cell fate determination during the development of the photoreceptors in the ommatidium of the eye and regulates proliferation during the development of the malpighian tubules by regulating the expression of cell cycle regulators.
In contrast to the related proteins COUP-TFI and COUP-TFII, the function of EAR-2 has not been well characterized. EAR-2 functions as a transcriptional repressor in vitro, inhibiting the transactivating ability of numerous genes including the thyroid hormone receptor (See Zhu, X. G. et al. (The orphan nuclear receptor Ear-2 is a negative coregulator for thyroid hormone nuclear receptor function. Mol Cell Biol 20, 2604-18. 2000)). Like many nuclear receptors, EAR-2 heterodimerizes with the retinoid X receptor-α (RXR-α), although the relevance of this interaction in EAR-2 function is unclear (See Ladias, (J. A. Convergence of multiple nuclear receptor signaling pathways onto the long terminal repeat of human immunodeficiency virus-1. J Biol Chem 269, 5944-51 1994)).
The role for EAR-2 in haematopoiesis has not been studied in vivo. A previous study has shown interaction of NR2F6 with the key haematopoietic transcription factor RUNX1 (also known as AML1) (See Ahn et al. (Negative regulation of granulocytic differentiation in the myeloid precursor cell line 32Dcl3 by ear-2, a mammalian homolog of Drosophila seven-up, and a chimeric leukemogenic gene, AML1/ETO. Proc Natl Acad Sci USA 95, 1812-7. 1998)). Targeted deletion of RUNX1, a component of the core binding factor complex, results in abrogation of definitive haematopoiesis and embryonic lethality and RUNX1 rearrangements result from several commonly seen chromosome translocations in acute leukemia. EAR-2 interacts physically with RUNX1 and represses its transcriptional activating ability in the murine myeloblast cell line 32Dcl3. The effect of NR2F6 in primary mouse or human bone marrow, let alone in vivo is unclear. EAR-2 is down regulated in 32Dcl3 cells induced to mature with G-CSF, and forced expression of the EAR-2 protein blocks 32Dcl3 differentiation.
The function of NR2F6 has not been well characterized. NR2F6 functions as a transcriptional repressor in vitro, inhibiting the transactivating ability of numerous proteins including the thyroid hormone receptor. Like many nuclear receptors, NR2F6 heterodimerizes with the retinoid X receptor-α (RXR-α), although the relevance of this interaction in NR2F6 function is unclear (See Ladias, J. A. (Convergence of multiple nuclear receptor signaling pathways onto the long terminal repeat of human immunodeficiency virus-1. J Biol Chem 269, 5944-51 1994)). A recent report describes the initial characterization of an NR2F6 deficient mouse generated by targeted disruption of the NR2F6 locus (See Warnecke, M et al. (Abnormal development of the locus coeruleus in Ear2(Nr2f6)-deficient mice impairs the functionality of the forebrain clock and affects nociception. Genes Dev 19, 614-25 2005)). NR2F6 deficient mice are viable and fertile, but show agenesis of the locus coeruleus, a midbrain nucleus that regulates circadian behaviour and nociception. In situ mRNA hybridization in NR2F6−/− animals places NR2F6 downstream of Mash1 and upstream of Phox2a and Phox2b in the specification of the locus coeruleus. Although NR2F6 expression is seen outside the central nervous system, this report contains no description of any phenotypic analysis outside the nervous system.